eLife digest

Diabetes mellitus is a disease that can lead to dangerously high blood sugar levels, causing numerous complications such as heart disease, glaucoma, skin disorders, kidney disease, and nerve damage. In healthy individuals, beta cells in the pancreas produce a hormone called insulin, which stimulates cells in the liver, muscles and fat to take up glucose from the blood. However, this process is disrupted in people with diabetes, who either have too few pancreatic beta cells (type 1 diabetes) or do not respond appropriately to insulin (type 2 diabetes).

All patients with type 1 diabetes, and some with type 2, must inject themselves regularly with insulin, but this does not always fully control the disease. Some type 1 patients have been successfully treated with beta cells transplanted from deceased donors, but there are not enough donor organs available for this to become routine. Thus, intensive efforts worldwide are focused on generating insulin-producing cells in the lab from human stem cells. However, the cells produced in this way can give rise to tumors.

Now, Lee et al. have shown that duct cells, which make up about 30% of the human pancreas, can be converted into cells capable of producing and secreting insulin. Ductal cells obtained from donor pancreases were first separated from the remaining tissue and grown in cell culture. Viruses were then used to introduce genes that reprogrammed the ductal cells so that they acquired the ability to make, process and store insulin, and to release it in response to glucose—hallmark features of functional beta cells.

As well as providing a potential source of cells for use in transplant or cell conversion therapies for diabetes, the ability to grow and maintain human pancreatic ductal cells in culture may make it easier to study other diseases that affect the pancreas, including pancreatitis, cystic fibrosis, and adenocarcinoma.

Introduction

The pancreas is a vital organ with exocrine and endocrine cell functions, and a root of lethal human diseases including diabetes mellitus, pancreatitis, and pancreatic ductal adenocarcinoma. Exocrine acinar cells produce digestive zymogens that are delivered to the intestines by a branching network of exocrine ductal cells that secrete bicarbonate and other products. Pancreatic endocrine functions derive from clusters of epithelial cells (islets of Langerhans) called α-, β-, δ-, and PP-cells that respectively synthesize, store, and secrete the hormones Glucagon, Insulin, Somatostatin, and Pancreatic polypeptide (Benitez et al., 2012). Insulin production by islet β-cells is highly regulated: key features of mature β-cells include preproinsulin (INS) transcription, proinsulin processing by endo- and exo-peptidases and storage of the proinsulin cleavage products insulin and C-peptide in dense core vesicles. Likewise, cardinal β-cell functions regulate insulin release in response to glucose and other secretagogues, including glucose sensing and metabolism through the enzyme glucokinase, and use of ATP-dependent potassium channels (KATP) and voltage-gated calcium channels to induce insulin exocytosis (reviewed in Suckale and Solimena, 2010). Deficiency or malfunctioning of β-cells produces impaired glucose regulation and diabetes mellitus, a disease with autoimmune (type 1, T1DM) and pandemic forms (type 2; Ashcroft and Rorsman, 2012). Thus, replacement or regeneration of functional human β-cells is an intensely-sought goal.

Human islet transplantation can be used to replace β-cell function in T1DM (reviewed in Vardanyan et al., 2010), but a shortage of donors currently precludes broad use of human pancreatic islets for β-cell replacement. Because of their expandability and multipotency, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been explored as sources of replacement insulin-producing cells (reviewed in Hebrok, 2012). However, directing the differentiation of these developmentally ‘primitive’ cells through multiple sequential fates into β-cell-like progeny that synthesize, process, store, and secrete insulin while lacking tumorigenic potential has challenged investigators worldwide (Fujikawa et al., 2005; McKnight et al., 2010; Cheng et al., 2012). Moreover, different hESC and iPSC cell lines exhibit significant variability during development into insulin-producing cells (Nostro and Keller, 2012). Recent work demonstrated that differentiated cell types in adult organs, including the mouse pancreas, can be experimentally ‘reprogrammed’ into progeny resembling islet cells, suggesting a new strategy for β-cell replacement (Vierbuchen and Wernig, 2011). For example, adult mouse pancreatic acinar cells can be converted into insulin-producing cells in vitro and in vivo (Minami et al., 2005; Zhou et al., 2008). However, little progress has been made in reprogramming primary human epithelial cells into different cell types, including conversion of pancreatic non-β-cells toward a human β-cell fate (Vierbuchen and Wernig, 2011). Thus, systems permitting expansion and genetic modulation of human pancreatic cells could powerfully influence studies of β-cell biology and replacement.

Clonal expansion and passaging of ductal spheres.

(A) Confocal images of 2-week-old spheres immunostained with KRT19, CD133, Ki-67, and Phospho-Histone H3 (all green). Note the apical localization of CD133. Scale bars, 50 µm. (B) Representative time-lapse images of sphere formation from single cell (arrowhead). Images taken every 12 hr for 9 days are shown. Arrows point a non-sphere forming cell used as a landmark. (C) Representative pictures of spheres after each passage. Scale bars, 100 µm. (D) Quantification of cell number in spheres after each indicated passage. Y axis represents fold increase of total cell numbers relative to the one measured in the first ‘generation’ of spheres (G1).

To assess whether sphere growth was achieved by cell proliferation or by other mechanisms like cell migration and aggregation, we analyzed spheres by immunostaining and time-lapse imaging. Immunohistochemistry revealed the proliferation marker Ki-67 in more than 25% of cells comprising 2-week-old spheres (Figure 2A, Figure 2—figure supplement 1B; labeling index 26.5 ± 5.1%), data further supported by detection of a second proliferation marker, phospho-histone H3 (Figure 2A). Time-lapse imaging revealed that spheres arose from single cells (Figure 2B), providing strong evidence that sphere formation resulted from CD133+ ductal cell proliferation, rather than through cell migration and aggregation. Enzymatic dispersion of 2-week-old G1 spheres and subsequent culture revealed that the spheres can be passaged up to seven generations (G7, 3 months) and that the total number of cells increased with each generation (Figure 2C,D, Figure 2—figure supplement 1C). After G7, ductal cell expansion was not achieved, and the spheres were not formed (Figure 2—figure supplement 1C and data not shown), supporting the view that ductal epithelial cells are not immortalized, and consistent with the origin of pancreatic cells from donors without neoplasia.

Immunostaining confirmed these qRT-PCR findings and demonstrated that only RFP+ cells produced by Ad-RFP-Neurog3 infection were immunostained with antibodies recognizing NEUROD1, NKX2.2, CHGA, SST or GHRL (Figure 3B,D, Figure 3—figure supplement 1). We also confirmed that no insulin-, glucagon- or PPY-positive cells were observed by immunostaining (data not shown). While only a subset of cells infected with Ad-RFP-Neurog3 (RFP+) expressed CHGA, we noted all GHRL+ or SST+ cells co-expressed CHGA (Figure 3D). Quantification of CHGA+ and hormone+ cells revealed that 30% of infected cells (RFP+) expressed CHGA. At least 45% of CHGA+ cells produced SST or GHRL, and less than 2% of CHGA+ cells expressed both hormones (Figure 3D,E). Thus, Neurog3 expression efficiently converted primary human ductal cells and cultured ductal epithelial spheres into hormone-expressing cells with cardinal features of endocrine pancreas.

In mice, Neurog3 gene dosage can determine commitment between exocrine and endocrine lineages in pancreas development (Wang et al., 2010). Therefore, we next assessed the possibility that the 70% of RFP+ cells infected by Ad-RFP-Neurog3 failing to express CHGA may have achieved inadequate levels of Neurog3 expression. We fractionated cells produced by Ad-RFP-Neurog3 infection by RFP intensity and measured mRNA expression of Neurog3, CHGA, SST and GHRL by qRT-PCR (Figure 3F,G). We found that cell fractions with the highest levels of RFP expression (‘P4 and P5’, Figure 3F) had the highest levels of mouse Neurog3 mRNA, and only these cell fractions produced mRNA encoding CHGA, SST or GHRL (Figure 3G). These data suggest that relatively high threshold levels of Neurog3 may be necessary and sufficient for directing endocrine differentiation of human pancreatic cells.

Conversion of pancreatic duct cells into progeny that produce, process, and store insulin

The transcription factors MafA, Neurog3, and Pdx1 (a combination hereafter summarized as ‘MNP’) were sufficient to convert adult mouse acinar cells into insulin-producing cells (IPCs: Zhou et al., 2008). We constructed three adenoviruses expressing MafA, Neurog3, or Pdx1 (see ‘Materials and methods’; Figure 4A), and infected cultured spheres with this MNP combination. Within 5 days after infection, we reproducibly detected INS mRNA induction but at extremely low levels relative to adult human islet controls (0.0035 ± 0.0012% of islet levels; Figure 4B). Thus, we sought additional factors and discovered that mRNA encoding PAX6, an important regulator of mouse pancreatic endocrine cell development (Sander et al., 1997), was induced by MNP to only 0.03% of levels in control islets (Figure 4—figure supplement 1A). When combined with MafA, Neurog3, and Pdx1 (encoded in four viruses, ‘4V’), Pax6 induced INS expression in primary CD133+ ductal cells or spheres by over 30-fold relative to MNP (Figure 4A,C,D). We observed ductal conversion to IPCs with four consecutive, independent donors (INS, Figure 4D). We also detected substantially increased expression of other islet endocrine markers, including SST, GCK, PCSK1, KCNJ11, and ABCC8 (Figure 4D). Immunohistochemical analyses demonstrated that the number of Insulin+ cells was increased by 18 to 20-fold in spheres transduced by the four factor combination (4V) compared to the MNP combination (Figure 4F,G). ELISA studies quantified and confirmed this increase of proinsulin levels, showing that the spheres derived from 4V exposure contained proinsulin levels that averaged 0.7% of those in human islets (Figure 5E). Systematic removal of individual factors from this four virus combination revealed that omission of Neurog3 prevented expression of INS, CHGA or SST (Figure 4E). Omission of virus expressing MafA or Pax6 from this combination significantly reduced INS expression (Figure 4E–G), whereas omission of virus expressing Pdx1 did not significantly decrease INS expression. Thus, Neurog3-mediated endocrine cell conversion is required for the production of IPCs as well as other hormone-producing cells from ductal spheres.

Regulated insulin C-peptide secretion by IPCs

Native islet β-cells depolarize and secrete insulin and C-peptide in response to glucose and other physiological or pharmacological stimuli, but reconstructing these hallmark functions in progeny of purified primary human non-β-cells has not been previously achieved during in vitro culture. Compared to baseline secretion in media with 0.1 mM glucose, IPCs increased insulin C-peptide secretion by 2.4-fold upon exposure to 2 mM glucose (Figure 5G). Similar to insulin release by human islet β-cells (Lupi et al., 1999), glucose stimulated the secretion of approximately 4% of total insulin C-peptide in IPCs (Figure 5—figure supplement 1G). This effect was blocked when the cells were incubated with glucose and Diazoxide, a drug that opens KATP channels and prevents glucose-stimulated insulin secretion (Figure 5G). However, unlike adult human islet β-cells, the release of insulin by IPCs was not further increased by 11 mM glucose. Islets from fetal or neonatal stages do not show elevated insulin secretion by high level glucose challenge (Rozzo et al., 2009), suggesting that IPCs are similar to immature islet β-cells and that further maturation is possible (Figure 5G). Calcium and voltage-dependent calcium channels are important regulators of normal insulin secretion after KATP channel-mediated membrane depolarization in β-cells (Henquin, 2005). When calcium was omitted in secretion buffer, C-peptide secretion stimulated by glucose was abolished, but restored upon calcium addition (Figure 5G). Insulin C-peptide release by cultured IPCs was also induced by the depolarizing agent potassium chloride (30 mM KCl), an effect reversed by a subsequent wash in media with 4.8 mM potassium ion (Figure 5G, figure 5—figure supplement 1H). Treatment with tolbutamide, a KATP channel blocker causing membrane depolarization, also stimulated insulin secretion by IPCs, an effect prevented by omission of calcium (Figure 5G). Together with data showing expression of key regulators of stimulus-secretion coupling, these findings provide strong evidence that IPCs produced by conversion and extended culture in our system develop regulated insulin secretion.

We examined the stability of the conversion of human ducts into IPCs by long-term transplantation of the converted spheres into specific transplantation sites of NOD scid gamma (NSG) mice (Figure 5—figure supplement 2; Supplementary file 1A). Human C-peptide was readily detected in kidney grafts harvested at specific times by immunostaining (8/12 cases, Figure 5—figure supplement 2A; Supplementary file 1A) and by ELISA (9/10 cases, Supplementary file 1A) without detectable tumor formation. This also included C-peptide+ IPCs left in the transplant location beyond 5 months (Figure 5—figure supplement 2A, d151), suggesting converted IPCs were stable. However, we observed that the total number of grafted C-peptide+ cells was drastically reduced within 2 weeks after transplantation, likely due to the apoptotic cell death. In three independent IPC transplants, however, we were able to detect circulating human insulin in the serum of host mice, and its level increased following intraperitoneal glucose challenge (Figure 5—figure supplement 2B; Supplementary file 1B). Therefore, these data suggest that despite extensive cell death in early stages of transplantation, IPCs can further mature in vivo and release increased levels of insulin in response to acute glucose challenge.

Discussion

Methods to regenerate lost or injured cells in diseases like diabetes mellitus are the focus of intensive investigations (McKnight et al., 2010; Benitez et al., 2012). Generation of insulin-producing cells from human stem cell lines like human ES cells (D’Amour et al., 2006; Kroon et al., 2008) is an important, and oft-cited ‘benchmark’, in efforts to achieve β-cell replacement. However, in these prior reports, progeny of human ES cells developed largely as poly-hormonal cells, most frequently expressing both glucagon and insulin. Moreover such hESC progeny failed to secrete insulin in response to glucose or other secretagogues unless transplanted as progenitors for >2 months in mice (Nostro and Keller, 2012). This transplant-based maturation strategy was complicated by tumor formation (Fujikawa et al., 2005). Thus, it has remained unknown whether human cells can develop solely in vitro to generate glucose-responsive insulin-secreting progeny without tumorigenicity. Our data indicate that in principal this can be achieved, using a small number of genes in sorted human pancreatic ductal cells that convert them toward an islet fate, including progeny that produce, store, and secrete insulin in response to glucose.

Conversion of mouse acinar cells into insulin-producing cells using adenoviral delivery of Neurog3, Pdx1, and MafA was previously reported (Zhou et al., 2008). However, it has remained unknown whether human pancreatic cells can be converted using transgenic methods toward a β-cell fate. We were unable to culture and expand primary human pancreatic acinar cells (Figure 1B and data not shown); moreover, we found that the combination of these three genes (MNP) was insufficient to reprogram primary or expanded human pancreatic ductal cells toward a β-cell fate, suggesting transgenic conversion may be restricted by species and cell type. Thus, we postulated that additional transcriptional regulators might be needed to promote human ductal conversion toward a β-cell fate. Like Neurog3, MafA, and Pdx1, the transcription factor Pax6 is expressed in both fetal and adult pancreas, and required to achieve appropriately high levels of Ins and Gcg expression in mouse islet cell development (Sander et al., 1997; Wang et al., 2009, 2010; Pan and Wright, 2011). Together with the other factors, we found that Pax6 significantly enhanced expression of β-cell markers during ductal reprogramming into β-cells, and was shown as an essential factor for this process. By systematic addition or omission of each transcription factor, we found PDX1 is not required for IPC formation. Thus, unlike mouse acinar cells (Zhou et al., 2008) and human hepatocytes (Sapir et al., 2005), human ductal cells do not require exogenous Pdx1 expression for conversion toward an endocrine fate, for reasons that remain unclear. Our findings are also consistent with recent reports that transgenic adult mouse ductal cells can generate endocrine cells in vivo (Al-Hasani et al., 2013).

We initially attempted to induce spontaneous differentiation of pancreatic ductal cells using systematic variations of culture conditions, but these efforts proved unsuccessful (J Lee, unpublished results). During pancreas development, Neurog3 level surges in a subset of pancreatic progenitors located in primitive ducts, inducing development of endocrine cell fates (Zhou et al., 2007; Miyatsuka et al., 2009). Therefore, based on this model, we attempted to mimic induction of Neurog3 in human ductal cells using adenoviral overexpression of Neurog3. We found that Neurog3 was necessary and sufficient for reprogramming human ductal cells, and that the level of ectopic Neurog3 mRNA expressed in ductal cells correlated well with the extent of endocrine reprogramming, including expression of islet hormones (Figure 3G). These findings are reminiscent of studies by Gu et al. showing that reduced Neurog3 gene dosage in mice leads to respecification of pancreatic endocrine progenitors into ductal and acinar cells (Wang et al., 2010). Thus, Neurog3 functions may be evolutionarily conserved in allocating cells toward an exocrine or endocrine fate (whether in development or experimental cell conversion) in a dosage-dependent manner. Consistent with prior work revealing that Neurog3 attenuates islet cell proliferation (Miyatsuka et al., 2011), we did not observe multiple rounds of cell division, an important prerequisite for some de-differentiation events (Hanna et al., 2009), during Neurog3-dependent cell conversion. Also, we observed Neurog3 induction alone can rapidly upregulate endocrine molecular signatures in cultured human ductal cells. Thus endocrine cell conversion described here may involve direct conversion of human ductal cells into endocrine cells, rather than de-differentiation, but additional studies are required to assess this possibility. Our findings, albeit with enforced transcription factor expression in adult cells, indicate that Neurog3 expression is sufficient to induce latent endocrine programs in human adult ductal cells, a capacity not yet clearly demonstrated, to our knowledge.

We demonstrated robust expansion of purified human ductal cells in 3-dimensional culture. The cells were clonally expanded and serially passaged up to seven generations over 3 months, achieving an increase in cell number calculated to be up to 3,200-fold. By contrast, in prior studies, the maximum duration of sustained culture achieved with primary human pancreatic ductal cells was 5 weeks (Trautmann et al., 1993; Bonner-Weir et al., 2000; Rescan et al., 2005; Hao et al., 2006; Yatoh et al., 2007; Hoesli et al., 2012). Moreover, cultured cells in spheres maintained cardinal features of primary pancreatic ducts such as apical-basal polarity and KRT19 expression up to seven generations (Figure 2—figure supplement 1D,E). Thus, features of our culture system may be useful for studying the genetics and biology of human ductal cells.

Prior studies have reported that duct-containing fractions from human adult pancreas can form insulin-producing cells in vitro (Bonner-Weir et al., 2000; Hao et al., 2006; Heremans et al., 2002; Noguchi et al., 2006; Koblas et al., 2008; Swales et al., 2012) or after xeno-transplantion in mice (Yatoh et al., 2007). However, the possibility of endocrine cell contamination in the initial ductal fraction or feeder/stromal cells used for co-culture was raised by the detection of mRNAs encoding islet cell hormones and other endocrine markers in these and other studies (Heremans et al., 2002; Gao et al., 2005). Therefore, it remained elusive whether human pancreatic ducts retained the potential to produce islet endocrine cells in adult. In this report, we used FACS to fractionate CD133+ ductal cells and used molecular and immunocytological studies to demonstrate complete elimination of cells expressing markers of differentiated endocrine cells (including islet hormones). Therefore, subsequent conversion of these cells into functional endocrine cells provided unequivocal evidence that endocrine cell-free human adult CD133+ ductal cell fraction can be converted into islet endocrine cells. Centroacinar cells are located at the junction of acini and tip of intercalated ducts (Cleveland et al., 2012) and their properties remain poorly understood. These cells express CD133 (Immervoll et al., 2008), raising the possibility that our fractionated CD133+ cells also include centroacinar cells. Based on their relative paucity in the pancreas, it is unlikely that centroacinar cells are the exclusive source of spheres within this CD133+ fraction, as more than 11% of CD133+ cells were capable of generating spheres (Figure 1B). However, because of difficulties performing lineage-tracing experiments with human samples, we cannot exclude the possibility that centroacinar cells may also contribute to the conversion into endocrine cell lineages.

While expression of Pax6 along with Neurog3, Pdx1 and MafA significantly enhanced expression of INS and other β-cell marker genes in converted ductal cells, this transcription factor combination alone was not sufficient to generate mature IPCs. We found that extending the culture period for 2 weeks after viral infection led to maturation of several hallmark β-cell functions, including expression of key β-cell factors, significant increases of INS mRNA and protein levels, proinsulin processing, dense-core granule formation, and Insulin secretion in response to glucose or other depolarizing stimuli. We tested four distinct culture media with or without serum for this extended culture, and all media permitted maturation of these β-cell properties in IPCs (Figure 5—figure supplement 1F and see ‘Materials and methods’), indicating that the duration of culture is a key variable for promoting β-cell maturation in vitro. After maturation, the spheres contained an average of 7% total insulin compared to human islet controls, and 7–11% of cells comprising these spheres produced insulin C-peptide. Thus, we calculate that each reprogrammed Insulin+ cell produced between 49 and 77% of insulin levels observed in native β-cell controls, a comparable level to the IPCs derived from human ES cells (D’Amour et al., 2006).

Is the capacity of human ductal cells to be converted toward endocrine islet fates unique? A prior study by Sapir et al. (2005) suggests that human hepatocytes may be induced to express insulin. However, the conversion toward an insulin-producing fate was comparatively poor; resulting cells produced about 10,000-fold lower insulin mRNA level than that of human islets, about 3–4 orders of magnitude lower than from conversion of pancreatic duct spheres. In addition, characteristic dense core vesicles in converted hepatocytes were not observed, indicating insufficient conversion towards β-cells. Here, we also assessed the endocrine potential of primary human dermal fibroblasts, cells successfully ‘reprogrammed’ toward many non-fibroblast fates, including induced pluripotent stem cells (Takahashi et al., 2007), but detected no clear evidence of conversion toward an endocrine or β-cell fate (Figure 5—figure supplement 4, see ‘Materials and methods’ for details). Thus, conversion of human adult duct spheres into cells that produce and secrete insulin is singularly robust. Moreover, unlike prior studies of human ES cells that have high variability among ES cell lines used (D’Amour et al., 2006; Kroon et al., 2008), we demonstrated conversion toward insulin+ fates by ductal cells from multiple unrelated donors, another feature of the robustness of our methods.

Expression of factors produced from viral transgenes persisted in Insulin+ cells for at least 5 months, evidenced by the GFP expression in transplanted insulin-producing cells (Figure 5—figure Supplement 2A and 3). The transgenes delivered by adenovirus do not generally persist in dividing cells (Zhou et al., 2008). We speculate that cell cycle arrest in Insulin+ cells may be induced by Neurog3 (Miyatsuka et al., 2011), thereby preventing dilution of viral transgene-encoded factors. Thus, further studies are needed to investigate how persistent expression of conversion factors like Neurog3 affects maintenance and maturation of endocrine phenotypes in converted cells. Survival of transplanted insulin-secreting cells produced from ductal cells was poor, and reduced yields following transplantation of ductal cells precluded physiological studies in mouse models of diabetes. Promoting survival of transplanted insulin-secreting cells is a general problem for transplant-based islet replacement approaches. Thus, studies of factors that enhance survival of Insulin+ ductal cell progeny are an important current focus.

In conclusion, our study provides unique evidence that primary human cells can generate progeny that produce, store and secrete insulin in response to glucose or depolarizing agents, the hallmark features of pancreatic β-cells. We also show that human pancreatic exocrine cells, like in mice (Zhou et al., 2008), can be converted by transgenes toward an endocrine islet-like cell fate. We speculate that gene-based strategies like those described here may be combined with other methods, including culture modulation by growth factors and small molecules (Warren et al., 2010), to optimize endocrine differentiation or conversion of diverse cellular sources to advance cell replacement for diabetes. We speculate that our cell culture system may also serve as the foundation to investigate the genetics and pathogenesis of diverse human diseases rooted in pancreatic ductal cells, including pancreatitis, cystic fibrosis, and adenocarcinoma.

Materials and methods

Cell preparation

Institutional review board approval for research use of human tissue was obtained from the Stanford University School of Medicine. Human islet-depleted cell fractions were obtained with appropriate consent from healthy, non-diabetic organ donors deceased due to acute traumatic or anoxic death by overnight shipping from the following facilities: Division of Transplantation (Massachusetts General Hospital, MA), UAB Islet Resource Facility (University of Alabama at Birmingham, AL), UCSF Diabetes Center (University of California, San Francisco, CA), Kidney/pancreas transplantation center (University of Pennsylvania, PA), Islet Core of the University of Pittsburgh (Pittsburgh, PA), and Human Islet Isolation Program (The Hospital of the University of Virginia, VA). Donor samples with the age range 16–63 years (mean 38.24 years) used for this study are listed in Table 1. On receipt, the cell fractions were washed with PBS and cultured with CMRL media (Mediatech, Inc, Manassas, VA) supplemented with 10% heat inactivated fetal bovine serum (FBS, HyClone, Logan, UT), 2 mM GlutaMax (Life Technologies, Grand Island, NY), 2 mM nicotinamide (prepared in PBS, Sigma, St.Louis, MO), and 100 U Penicillin and 100 µg Streptomycin (Pen/Strep, Life Technologies) in a non-coated culture dish at 25.5°C in 5% CO2 until use. For dissociation, the cell pellet was washed with PBS, trypsinized with 0.05% Trypsin-EDTA solution (Life Technologies) for 5 min, and quenched with 5 vol of FACS buffer (10 mM EGTA, 2% FBS in PBS). Cells were collected by centrifugation and further digested in 1 U/ml dispase solution (Life Technologies) containing 0.1 mg/ml DNaseI in PBS on a nutating mixer at 37°C for 30 min. PBS washing was performed after each enzymatic digestion step. After centrifugation, the cell pellet was resuspended in FACS buffer and passed through a 40-µm-cell strainer. Cell viability and number were assessed using a Vi-Cell analyzer (Beckman Coulter, Fullerton, CA) and the samples exceeding 70% cell viability were used for subsequent antibody staining for FACS.

Cell sorting and culture

Dissociated cells were stained with biotin-conjugated CD133 antibodies (clone AC133 and 293C3, Miltenyi Biotec, Auburn, CA) and then Allophycocyanin-conjugated Streptavidin (eBioscience, San Diego, CA) for 15 min, each at room temperature. Cell pellets were collected by centrifugation and washed with PBS after each staining steps. Propidium Iodide (Life Technologies) staining was used to exclude dead cells. The cells were sorted using a FACSAria II (BD Biosciences, Bedford, MA) and collected in 100% FBS, washed with PBS twice, and resuspended in ice-cold Advanced DMEM/F-12 media (Life Technologies) at a density of 8000 cells/µl. The average percentage of CD133+ fraction was 32.73% (n = 32). 50 µl of growth factor-reduced Matrigel (BD Biosciences) was then added to 30 µl cell suspension and the mixture was placed around the bottom rim of each well. After solidification at 37°C for 60 min, each well was overlaid with 500 µl of modified crypt culture media (Sato et al., 2009) comprised of Advanced DMEM/F-12 media supplemented with recombinant human (rh) EGF (50 ng/ml, Sigma), rhR-spondin I (500 ng/ml, R&D systems, Minneapolis, MN), rhFGF10 (50 ng/ml, R&D systems), recombinant mouse Noggin (100 ng/ml, R&D systems), 10 mM Nicotinamide in PBS, and Pen/Strep. The media was changed twice weekly. The spheres were harvested after 2 to 3 weeks for passaging or viral infection. Static and time-lapse images of sphere growth were collected using Zeiss Axiovert 200 inverted microscope and Zeiss Observer.Z1 equipped with a temperature- and CO2-controlled chamber using Axiovision (Carl Zeiss, Germany) and MetaMorph (Molecular Devices, Sunnyvale, CA) softwares, respectively. For harvesting spheres, 500 µl of 2 U/ml dispase (Life Technologies) solution containing 0.1 mg/ml DNaseI in PBS was added in each well and the Matrigel was mechanically disrupted by pipetting and incubated at 37°C for 45 min. The released spheres were collected, washed twice with PBS and used for subsequent applications. For passaging spheres, the harvested spheres were trypsinized at 37°C for 5 min followed by quenching with FBS. The dispersed cells were then used for cell counting with a hemocytometer or were plated as described above.

Construction of adenoviral vectors

Ad-eGFP and Ad-RFP control adenoviruses were purchased from Vector Biolabs (Philadelphia, PA). Ad-MafA and Ad-Neurog3-IRES-eGFP were described previously (Tashiro et al., 1999). To construct Ad-RFP-Neurog3 and Ad-RFP-Pdx1 adenoviruses, mouse cDNAs for Neurog3 (BC104326) and Pdx1 (BC103581) were purchased from Open Biosystems (Lafayette, CO) and the inserts were obtained by restriction enzyme digestion with EcoR V/BamH I and EcoR V/Msc I, respectively. The inserts were then subcloned into multiple cloning sites of Dual-RFP-CCM shuttle vector (Vector Biolabs) and adenoviruses were constructed by Vector Biolabs. For Ad-eGFP-M6P, human MAFA cDNA (gift from M German), PDX1 (NM_000209; GeneCopoeia, Rockville, MD), and PAX6 (BC011953; Open Biosystems) were used for PCR amplification with the primers shown in Supplementary file 1C to add T2A, P2A, restriction enzyme sites, and/or tagging proteins (Figure 5—figure supplement 5). A fused construct of MAFA-T2A-PAX6 was generated by PCR with MAFA and PAX6 PCR amplicons as templates. Similarly, PCR products for PAX6 and PDX1 were used to construct PAX6-P2A-PDX1. Next, MAFA-T2A-PAX6, PAX6-P2A-PDX1, and pDual-GFP-CCM vector (Vector Biolabs) were cut with BglII/PstI, PstI/EcoRI, and BglII/EcoRI, respectively, and ligated with NEB quick ligation kit (New England Biolabs, Ipswich, MA) followed by transformation of TOP10 chemically competent cells (Invitrogen, Carlsbad, CA). The construct was then used for generating adenoviruses by Vector Biolabs.

cDNA preparation and qRT-PCR analyses

Total RNA was prepared from sorted cells or cultured spheres with QIAGEN RNeasy micro kit (QIAGEN Sciences, MD), and used for cDNA synthesis using QIAGEN Omniscript RT kit (QIAGEN), according to the manufacturer’s protocol. Relative mRNA level was measured by qRT-PCR of each cDNA in duplicate with gene-specific probe sets (Applied Biosystems, Foster City, CA) with TaqMan Universal PCR Master Mix (Applied Biosystems) and the ABI Prism 7500 detection system (Applied Biosystems). Normalizations across samples were performed using β-actin primers. Information of the primer and probe sets is available upon request.

Electron microscopy

The samples were fixed in Karnovsky’s fixative: 2% Glutaraldehyde (EMS Cat# 16000) and 4% Paraformaldehyde (EMS; Electron Microscopy Sciences, Hatfield, PA) in 0.1 M Sodium Cacodylate (EMS) pH 7.4 for 1 hr at RT then cut, post fixed in 1% Osmium tetroxide (EMS) for 1 hr at RT, washed three times with ultrafiltered water, then en bloc stained for 2 hr at RT or moved to 4°C overnight. The samples were then dehydrated in a series of ethanol washes for 15 min each at 4°C beginning at 50%, 70%, 95%, where the samples are then allowed to rise to RT, changed to 100% two times, followed by Acetonitrile for 15 min. The samples are infiltrated with EMbed-812 resin (EMS) mixed 1:1 with Acetonitrile for 2 hr followed by two parts EMbed-812 to 1 part Acetonitrile for 2 hr. The samples were then placed into EMbed-812 for 2 hr and then placed into molds, and resin filled gelatin capsules with labels were orientated over the cells of interest and placed into 65°C oven overnight. Sections were taken between 75 and 90 nm on a Leica Ultracut S (Leica, Wetzlar, Germany), picked up on formvar/Carbon coated slot grids (EMS Cat#FCF2010-Cu) or 100 mesh Cu grids (EMS). Grids were contrast stained for 15 min in 1:1 saturated UrAcetate (∼7.7%) to 100% ethanol followed by staining in 0.2% lead citrate for 3 to 4 min. JEOL JEM-1400 TEM was used to observe at 120 kV and photos were taken using a Gatan Orius digital camera.

C-peptide secretion and content measurement

C-peptide secretion assay and content measurement were performed as described previously with minor modification (Chen et al., 2001). Briefly for secretion assay, media was replaced a day before assay was performed. On the day, each well with matrigel-embedded spheres was incubated with fresh media for 2 hr, washed twice with plain Krebs-Ringer bicarbonate buffer (KRBB), and incubated twice with plain KRBB for 1 hr each for thorough washing. Next, the spheres were incubated consecutively with 400 µl KRBB containing indicated concentrations of glucose (Sigma) with or without 0.5 mM Diazoxide (Sigma), KCl (30 mM, Sigma), or Tolbutamide (0.2 mM, Sigma) for 2 hr each. KRBB without Calcium (No Ca++) was prepared by omission of CaCl2 and addition of 1 mM EGTA (Sigma). Secreted C-peptide level was measured with Human Ultrasensitive C-peptide ELISA kit (Mercodia). For C-peptide content measurement, the spheres were harvested in 1.5 ml microfuge tube, washed with PBS, resuspended with 300 µl of ice-cold TE/BSA buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1% wt/vol BSA, pH 7.0), and sonicated with Bioruptor Sonicator (Diagenode, Denville, NJ). Half of the lysate was used for genomic DNA isolation and quantification with Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). Same volume of acid alcohol (75% vol/vol ethanol, 2% vol/vol concentrated HCl, 23% vol/vol H2O) was added to the rest of lysate to extract C-peptide by rocking overnight at 4°C. The extract was then neutralized with 10 vol of PBS and used for C-peptide ELISA.

Transplantation

Transplantation in kidney capsule, epididymal fat pad (EFP), or in the liver by portal vein injection was performed as previously described (Kroon et al., 2008; Alipio et al., 2010; Wang et al., 2011). For transplantation in kidney or EFP, converted spheres with or without extended culture were harvested and mixed with or without mouse embryonic fibroblasts (Supplementary file 1A). The spheres were then mixed with matrigel to make a final volume of 10 µl for kidney transplantation or overlayed on pre-wet gelfoam for EFP transplantation. For liver transplantation, single cells produced by trypsinization of harvested spheres were resuspended in 100 µl PBS and injected into the portal vein with a 27 G needle. All animal experiments and methods were approved by the Institutional Animal Care and Use Committee (IACUC) of Stanford University.

In vivo glucose-stimulated insulin secretion assay

Secretion of human Insulin or C-peptide by glucose injection was measured as previously described (Kroon et al., 2008). Briefly, transplanted mice were fasted overnight (14–16 hr) and 120 µl of blood was collected from tail into Microvette CB300LH (Sarstedt, Germany) to prepare 50 µl of serum. 3 g/kg glucose was then injected and blood was collected again 30 min after glucose administration. Secreted C-peptide or insulin level was measured with Human Ultrasensitive C-peptide or Insulin ELISA kits (Mercodia).

Human adult dermal fibroblast culture and conversion assay

Human adult dermal fibroblasts (Coriell Institute for Medical Research, Camden, New Jersey, USA) were cultured and maintained as described previously (Yoo et al., 2011). The cells were either trypsinized for suspension infection (as was described above for ductal spheres) or infected as adherent cells in six-well plates by direct addition of virus into the culture medium, with Ad-eGFP (GFP) or Ad-eGFP-M6P and Ad-Neurog3-IRES-eGFP (4TFM). The same MOIs used for ductal sphere infection were also used. The suspension-infected cells were harvested the following day and embedded in Matrigel as described above for infected ductal spheres. The culture was maintained for additional 18 days to match the duration of infected ductal sphere maturation. The infected adherent cells were cultured with virus for 48 hr and the media was replaced. The culture was maintained for additional 10 days, passaged in 1:3 ratio due to confluency, re-plated, and cultured additional 7 days to match the duration of infected ductal sphere maturation. In both cases, media was replaced every other day. Three independent experiments were performed for both conditions and each experiment at least in duplicates. RNA isolation, cDNA preparation, and qRT-PCR were performed with primers specific to human INS, CHGA, and β-actin as described above.

Decision letter

Janet Rossant

Reviewing Editor; University of Toronto, Canada

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]

Thank you for choosing to send your work entitled “Expansion and Conversion of Human Pancreatic Ductal Cells into Insulin-Secreting Endocrine Cells” for consideration at eLife. Your full submission has been peer reviewed by one of our Senior editors, Janet Rossant, and two other reviewers, and the decision was reached after discussions between the reviewers. We regret to inform you that your work will not be considered further for publication at this point.

The reviewers and the Senior editor have had an extensive online discussion about your paper, after exchanging the reviews. While they all feel that the experiments are carefully carried out, and the data presented are robust, in the end they were not convinced that the study as a whole provided a major step forward in the drive towards generating functional beta cells from other cell types. It was noted that you have not demonstrated whether the use of ductal cells (CD133+ cells) is advantageous over the use of other cell types for transdifferentiation. What would happen if the same factors were used to reprogram other cell types, even non-pancreatic cells, such as fibroblasts? The fact that ductal cells can respond to exogenous transcription factors does not directly demonstrate that these cells have latent potential to form beta cells, as claimed in the Abstract. It was also noted that the beta cells produced are not apparently fully mature and, therefore, the long-term significance of this approach for human therapy is unclear. The ability to grow human ductal cells in vitro is interesting and a further analysis of the non-transduced cells to respond to external signals and undergo differentiation into different cell types would be interesting.

Given these major concerns and the amount of extra work that would be needed to address them, the decision is to reject the manuscript at this time. A majorly enhanced experimental study including assessing whether ductal cells are uniquely responsive to these inducing factors, better characterization of the cells produced, and a further analysis of the unmanipulated ductal cells could form the basis of a new submission at a later date. The major points from the full reviews are provided below.

Reviewer #1:

In this manuscript the authors show that they can sort human cadaveric pancreas tissue with antibody to CD133 and that this enriches for pancreatic ductal epithelium. They then show that they can generate clonal spheres from these cells that can be passaged several generations in culture. They then infect these cultures with adenovirus expressing first neurogenin and then additional sets of beta cell inducing transcription factors and show that they can induce expression of endocrine phenotypes in the spheres and that a combination of 4 factors generates insulin-producing cells that show some degree of glucose regulation. The final most successful converted cells express 7% of the levels of insulin found in adult islets, but when transplanted to the kidney capsule in mice, they were able to detect some human insulin in serum. However, in these grafts cell survival was poor, so they were unable to test the ability of the cells to rescue diabetic mice.

This study is well performed and does indicate that ductal cells may be responsive to exogenous transcription factors that can drive towards an islet cell fate.

Major comments:

1) It is not clear whether conversion to islet cells is a unique property of ductal cells or whether other pancreatic cells or other cell types could respond in the same way. Other groups have suggested that hepatocytes can be transdifferentiated to insulin-producing cells- is this system more or less effective?

2) The final cocktail of transcription factors is stated to produce monohormonal insulin-producing cells, but this is not directly shown in the figures. This is an important point because many other differentiation assays generate fetal-like polyhormonal cells that cannot respond to glucose in the manner of adult beta cells. The cells produced here are not fully functional adult-type cells.

3) How long does expression of the exogenous factors persist? Is it required for ongoing maintenance of the cells or can you demonstrate independence of the exogenous factors?

4) How sure are they that the starting population is pure ductal cells, given that CD133 is not exclusively expressed in ductal epithelium in the pancreas? Can they double sort with a general epithelial marker to further purify the population, given that CD133 only enriches 4-fold for sphere-forming cells?

Reviewer #2:

In this study the authors describe a method to isolate and expand human ductal cells using an antibody against CD133. Using culture conditions similar to those described by Dr. Hans Clevers (Sato et al.), they were able to culture CD133+ cells as epithelial spheres that maintain a ductal phenotype and lack acinar and endocrine markers. Furthermore, the authors were able to reprogram the CD133-enriched population to endocrine cells by infecting isolated CD133+ cells and/or CD133+ -derived spheres with adenoviruses expressing ngn3, MAFA, PAX6 and PDX1. On average the authors are able to generate 10% insulin+ cells that resemble fetal beta cells, as they secrete insulin in response to low level of glucose (2mM), but fail to respond to higher glucose concentration (11mM). Following transplantation of the reprogrammed spheres into NSG mice, they observed that survival of the graft after transplantation was poor. However, they were able to detect human insulin in the serum of the host mice and showed that insulin levels increased after glucose challenge, suggesting in vivo maturation, albeit few mice were analysed. In general this work is very well done, with convincing images and clear data. There are only some minor points that need to be clarified.

Minor comments:

1) As CD133 is detected in centroacinar cells as well as in ductal cells (Immervoll et al JHC 2011), the authors should include additional acinar markers in their expression profile (Fig 1 D) to exclude acinar contamination.

2) Please include the average percentage of CD133+ cells detected in human pancreas and the purity of the sorted populations.

4) The authors state “Time-lapse imaging revealed that spheres arose from single isolated CD133+ ductal cells”. However, this statement is not accurate unless the purity of the sort was 100%.

5) Does the percentage of CD133+ cells decrease in culture? What is the percentage of CD133 after 3 months in culture?

6) The authors state that Insulin+ IPCs did not express other islet hormones, the authors should include co-staining of c-peptide with GCG, PP and Ghrelin in Figure 5D.

Reviewer #3:

The claim that adult human pancreatic duct cells have a latent capacity for endocrine differentiation is correct, but only in this context of extremely strong transcription-factor-based enforced differentiation. I always wonder what MNP6 would do to a non-pancreatic cell type, and therefore if this effect is a specific latency of pancreatic duct cells or not (the paragraph starting ‘The transcription factors MafA, Neurog3 and Pdx1…’ is more important as a result if there is something these cells can do that is not ever seen with the 4-factor combination MNP6 (4TF) or 4TFM).

Essential is the claim that the insulin-producing cells are mono-hormone-positive, but this is not shown in the paper. The authors refer to the Figure 5D as the one showing no double-positives, but no Gcg, PP, or Ghrl are shown here.

Some more clarity on this aspect seems critical. Assays for Gcg and other hormones are either referred to as data not shown, or this aspect is stated but the figure does not have the supporting data. Gcg immunodetection was done on cells from N alone? Gcg, PP were tested on the 4-factor combinations?

Is Pax6 already expressed in the MNP-adenovirus cultures? If so, why is more needed?

The part on “We sought new methods to mature the cells” (my words) reads strangely. It's the same method, just extending the time frame, I believe, and I would simplify this text. Another comment here is that we revert to the MNP6 mixture (4TF), having just been told that Pdx1 can be removed without impact - why?

Figure 2C–figure supplement 1 has an incorrect y-axis. 10,000 percent to the log(base10) is 4, correct. This graph needs altering – why not just “fold” for cell number? Related to this, Discussion asserts 3,000-fold, but this is just once? Up to 3,000-fold, and more typically xx-fold? Why do the cultures suddenly go bad at G7 (see text) – what happens – sudden apoptosis? #48 seems to be continuing even at G7 – please amend this text.

Does the 3-factor system (MNP) work in the authors' hands on mouse acini? And/or duct?

Does the CD133 separation method include centroacinar cells (CAC) or not? What is the photograph in Figure 1 – ‘tip’ of duct dives out of plane of section before it is reached, and therefore the CAC cannot be seen in this panel? CAC could have a specific latency not seen in duct cells.

The idea that lineage-tracing methods are hard to apply in human cells should be stated, as everything else hinges on numerical arguments on cleanliness of cell separations, etc.

chgA is an endocrine differentiation marker. This statement is meant to indicate that full-blown differentiation to the hormone-expressing state requires a substantial pulse of Ngn3. What about other hormones, even indicating non-pancreatic cell types?

A major point is the longevity of the pulse of Ngn3 and the other factors achieved with these methods, and the detection of a program that runs from the endogenous loci with or without the continuous presence of N, MNP, MNP6 (4TF, 4TFM). Would this method pulse the cells or not, and is continuous presence of some of the factors preventing their full differentiation to the most mature state?

Title of the section starting ‘Although ELISA studies readily detected Proinsulin production by IPCs…’ reads odd to me: ‘genetic conversion’ reads as if the genome of the adult duct cells is being altered in some manner.

Systematic removal of factors from MNP6 mixtures: Why can Pdx1 be removed without any impact?

Various ‘obvious’ markers were not tested, or the manuscript is incorrect in not showing such ‘easily pointed out’ questions. Pdx1 is produced, by immunofluorescence assay, within these induced beta cells? To normal levels? MafA/B, etc? Nkx6.1 was assessed, but the ‘dogmatic’ mature β-cell TF list should be addressed, at least.

It does seem difficult to follow 4V, 4TF, 4TFM, MNP. Seems as if there is a mixed descriptor used for the same manipulation, at least sometimes; I suggest simply checking for a way of making the text fully consistent throughout.

Author response

We are grateful that the initial review has provided such useful suggestions for improving our study, and that the overall view of the experimental strategy, concepts, and data quality were so positive. We have provided experimental data, most of it new, to address all the remaining major concerns summarized in the decision letter, including (1) new data with human fibroblasts to assess whether primary human pancreatic ductal cells are uniquely responsive to conversion conditions identified here, (2) better characterization of the cells produced, especially studies to establish their mono-hormonal development, and (3) further analysis of unmanipulated ductal cells. We trust our responses to these requests for additional data meet or exceed the reviewers' expectations.

In addition to the detailed responses to specific points below, we would like to address the general point about the importance of the findings presented here. We thank the reviewers for the opportunity to clarify why our work “represents a major step forward in the drive toward generating functional beta cells from other cell types”.

An important, and oft-cited ‘benchmark’, in this effort is production of insulin-producing cells from stem cell lines like human ES cells (D’Amour et al., 2006; Kroon et al., 2008 and subsequent work). However, these cells develop largely as poly-hormonal cells (most frequently expressing both glucagon and insulin) and fail to secrete insulin in response to glucose or other secretagogues unless transplanted for >2 months in mice (reviewed in Nostro and Keller, 2012). Thus, it has remained unknown whether human cells can develop solely in vitro to generate glucose-responsive insulin-secreting progeny and without tumorigenicity. Our data indicate that in principle this can be achieved. Moreover, the depth and quality of phenotyping we perform to characterize achievement of beta-cell fate (in our view) is unmatched by any other prior study, with inclusion of ultrastructural, in vitro, and in vivo secretion data.

A second as yet unanswered question in the field is whether human pancreatic cells can be converted using transgenic methods toward a ?-cell fate. The Melton group (Zhou et al., 2008) established that this was possible in mice using viral methods in exocrine acinar cells, but since that seminal study the field has yearned to know how relevant this strategy might be for human cells, especially human pancreatic cells. We feel that our work unequivocally establishes that human pancreatic ductal exocrine cells have latent potential to produce functional endocrine islet-like cells.

Third, there is a longstanding debate about the ability of pancreatic ductal cells to generate endocrine progeny, with the majority of experimental assessment of this important question previously performed in transgenic mice. Thus, we feel our work provides important unique evidence that pancreatic ductal (or centroacinar) cells have such endocrine potential. Moreover, our work addresses a gap in knowledge in our field about (and a new system for evaluating) the potential of human ductal cells for alternate fates.

The identification of a new cellular source and genetic strategy for generating progeny with features of functional ?-cells should advance the long-term development of cell replacement options in type 1 diabetes. Thus, we feel quite strongly that our work provides a major contribution by addressing and answering multiple outstanding questions in diabetes research and pancreas biology. We hope that our revision clarifies this contribution and is now deemed worthy of publication in eLife.

Reviewer #1:

1) It is not clear whether conversion to islet cells is a unique property of ductal cells or whether other pancreatic cells or other cell types could respond in the same way. Other groups have suggested that hepatocytes can be transdifferentiated to insulin-producing cells – is this system more or less effective?

We fully agree with this reviewer and the similar comment from Reviewer #3 that additional assessment of other human cells would enhance the impact and conclusions from our study. Thus, we attempted to convert human adult dermal fibroblasts using our conversion methods; these cells have been previously used successfully in other reprogramming experiments (Yoo et al., 2011). With the same 4TFM condition and Insulin or ChromograninA (ChgA) mRNA levels as readouts, we detected no clear evidence of conversion toward an endocrine or β-cell fate (n ≥ 6 from 3 independent experiments: Figure 5–figure supplement 4; see Materials and Methods for details). This indicates that human adult dermal fibroblasts have little to no ?-cell conversion potential. Despite repeated attempts, we were unable to assess the conversion property of human acinar cells due to their inability to grow in culture (Figure 1B CD133-negative population), consistent with other reports that primary acinar cells are difficult to culture. Thus, although we are unable to test all human cell types in this way, our work suggests that primary adult pancreatic ductal cells have a special latency for conversion toward an endocrine islet and beta cell-like fate.

To our knowledge, work by Sapir and colleagues (Sapir et al., 2005) is the only report describing the use of human primary hepatocytes for β-cell transdifferentiation. In this report, however, the conversion toward an insulin-producing fate was comparatively poor; resulting cells produced about 10,000-fold lower insulin mRNA level than that of human islets, about 3–4 orders of magnitude lower than from conversion of pancreatic duct spheres. In addition, characteristic dense core vesicles in converted hepatocytes were not observed, indicating insufficient conversion towards ?-cells. In our report, we have presented evidence for acquisition of several characteristics of insulin-producing cells by converted duct spheres, including development of characteristic dense core vesicles, high Insulin content (estimated 49–77% total insulin in each insulin-producing cell compared to human islets), absence of glucagon or other islet hormone co-expression, modest glucose sensing and insulin secretion capacity, and other features detailed in our results.

These features also distinguish our work from studies reporting insulin-producing cells produced from in vitro differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In our opinion, the qualitative and quantitative level of beta-cell phenotypes achieved by genetically-directed duct sphere conversion is higher than that achieved by prior hESC or iPSC studies in vitro. Moreover, our findings suggest that genetic methods may enhance beta-cell development from such cell lines

In sum, we believe conversion of human adult duct spheres is by far the most robust method yet reported for generating insulin-producing cells from human primary cells. We incorporated these points in the revised manuscript to clearly demonstrate the advantage of ductal cell use in human. We thank the reviewer for pointing this out.

2) The final cocktail of transcription factors is stated to produce monohormonal insulin-producing cells, but this is not directly shown in the figures. This is an important point because many other differentiation assays generate fetal-like polyhormonal cells that cannot respond to glucose in the manner of adult beta cells. The cells produced here are not fully functional adult-type cells.

We used immunochemistry to detect co-expression of C-peptide with GCG, PPY, and GHRL, and included such GCG and GHRL co-staining images in a new Figure 5D. We found no PPY-positive cells from 7,487 GFP-positive cells we screened from two different samples. We found three GCG- positive cells out of 4,460 GFP-positive cells screened and none were co-labeled with C-peptide.

3) How long does expression of the exogenous factors persist? Is it required for ongoing maintenance of the cells or can you demonstrate independence of the exogenous factors?

Expression of the exogenous factors persisted after two weeks of maturation period (Figure 5–figure supplement 3) and at least 5 months, evidenced by the GFP-positive insulin-producing cells in transplanted mice (Figure 5–figure supplement 2A). Transgenes delivered by adenovirus do not generally persist long term (Zhou et al., 2008), especially in dividing cells. Persistent transgene expression noted in our study likely results from the known ability of Neurog3 to induce cell cycle exit (Miyatsuka et al., 2011), thereby preventing dilution of transgene-encoded factors by cell division. This precluded tests to demonstrate independence of the transgene-encoded factors for ongoing maintenance of the converted-cell phenotypes observed. We clarified this point in our revised manuscript.

4) How sure are they that the starting population is pure ductal cells, given that CD133 is not exclusively expressed in ductal epithelium in the pancreas?

In human pancreas, CD133 is expressed exclusively in ductal cells and centroacinar cells, but not other cell types in pancreas, including acinar or islet endocrine cells. This has been shown by us (Figure 1C, Figure 1–figure supplement 1B) and by others (Immervoll et al., BMC cancer, 2008; Lardon et al., Pancreas, 2008). In addition, we assessed the purity of the ductal cell fraction collected by qRT-PCR and immunostaining of CD133+ “sorted” cells (Figure 1D–F). Thus, we are confident that CD133-based sorting can efficiently eliminate contaminating native islet endocrine cells, permitting conclusions about conversion toward endocrine cell fate. We understand that centroacinar cells may still be included in our CD133+ fraction. Therefore, we modified the phrase “purify ductal cells” to “fractionate ductal cells” in our revised manuscript.

Can they double sort with a general epithelial marker to further purify the population, given that CD133 only enriches 4-fold for sphere-forming cells?

Ductal cells already constitute large portion in unsorted cell population, ranging from 30% to 40% (Bouwens and Pipeleers, 1998). Therefore, an average of 4-fold enrichment is expected even by achieving pure ductal cell isolations. Supporting this is our finding that the CD133-negative population is completely devoid of the sphere-forming cells (Figure 1B).

Reviewer #2:

In general this work is very well done, with convincing images and clear data.

We thank the reviewer for this positive assessment.

1) As CD133 is detected in centroacinar cells as well as in ductal cells (Immervoll et al JHC 2011), the authors should include additional acinar markers in their expression profile (Fig 1 D) to exclude acinar contamination.

As requested, we performed qPCR with additional acinar marker carboxyl ester lipase (CEL) and included this data in Figure 1–figure supplement 1C.

2) Please include the average percentage of CD133+cells detected in human pancreas and the purity of the sorted populations.

As suggested, we calculated the average percentage of CD133+ fraction as 32.73% (n=32). We included this in our revised manuscript. To assess the purity of the sorted populations, we included qRT-PCR and immunostaining data of the “sorted” CD133+ and CD133-negative population with various markers in Figure 1D–F.

4) The authors state “Time-lapse imaging revealed that spheres arose from single isolated CD133+ductal cells”. However, this statement is not accurate unless the purity of the sort was 100%.

We agree with this point. To eliminate any possible confusion, we modified the sentence to “Time-lapse imaging revealed that spheres arose from single cells.”

5) Does the percentage of CD133+cells decrease in culture? What is the percentage of CD133 after 3 months in culture?

We used immunochemistry to quantify CD133 expression in cells comprising G1 and G7 spheres and quantified CD133+ cells. 95.0 ± 3.0% and 98.1 ± 0.58% cells were positive for CD133 in G1 and G7 spheres, respectively (n=6 sections each, ≥ 1300 cells counted). Rare CD133-negative cells are likely due to the exclusion of cell apical regions during tissue section, where CD133 expression is localized. We included this data in Figure 2–figure supplement 1D and E, and evaluated these results in our discussion.

6) The authors state that Insulin+IPCs did not express other islet hormones, the authors should include co-staining of c-peptide with GCG, PP and Ghrelin inFigure 5D.

We agree and now include appropriate images of the requested co-immunostaining results. We performed co-staining of C-peptide with GCG, PPY, and GHRL, and included GCG and GHRL co-staining images in a new Figure 5D. We found no PPY-positive cells from 7,487 GFP-positive cells we screened from two different samples. We found three GCG-positive cells out of 4,460 GFP-positive cells screened and none were co-labeled with C-peptide.

Reviewer #3:

The claim that adult human pancreatic duct cells have a latent capacity for endocrine differentiation is correct, but only in this context of extremely strong transcription-factor-based enforced differentiation.

We did not know if this comment required a response but for completeness, we included the following paragraph in our manuscript discussion.

I always wonder what MNP6 would do to a non-pancreatic cell type, and therefore if this effect is a specific latency of pancreatic duct cells or not (the paragraph starting ‘The transcription factors MafA, Neurog3 and Pdx1…’ is more important as a result if there is something these cells can do that is not ever seen with the 4-factor combination MNP6 (4TF) or 4TFM).

Please see our response to the similar comment (#1) from the first reviewer.

In addition, in every experiment and analysis we performed, we included spheres (derived from CD133+ pancreatic ducts) infected with control viruses (Ad-RFP or Ad-GFP) but otherwise identical conditions as with N, 4TF or 4TFM spheres. We did not detect evidence of endocrine cell conversion by qRT-PCR, immunohistochemistry, and Insulin C-peptide ELISA (data from these controls are all included and labelled appropriate a “N”, “4TF” or “4TFM”). Therefore, we concluded that spontaneous conversion does not occur at detectable frequency.

Essential is the claim that the insulin-producing cells are mono-hormone-positive, but this is not shown in the paper. The authors refer to theFigure 5Das the one showing no double-positivities, but no Gcg, PP, or Ghrl are shown here. Some more clarity on this aspect seems critical. Assays for Gcg and other hormones are either referred to as data not shown, or this aspect is stated but the figure does not have the supporting data. Gcg immunodetection was done on cells from N alone? Gcg, PP were tested on the 4-factor combinations?

We agree that the claim of mono-hormonal development is important to document. Please see also our response to a similar comment (#2) from the first reviewer. We were unable to detect mRNA or protein for GCG and PPY in Ad-RFP-Neurog3 infected spheres by qRT-PCR, now stated in the manuscript and in a revised Figure 4–figure supplement 1A. For 4TFM spheres, we performed co- staining of C-peptide with GCG, PPY, and Ghrelin, and included GCG and GHRL co-staining images in a new Figure 5D. We found no PPY-positive cells from 7,487 GFP-positive cells we screened from two different samples. We found three GCG-positive cells out of 4,460 GFP-positive cells screened and none were co-labeled with C-peptide. Thus we feel our claim of mono-hormonal insulin+ cells is well documented.

We initially attempted to induce spontaneous differentiation of pancreatic ductal cells using systematic variations of culture conditions, but these efforts proved unsuccessful. This motivated us to induce Neurog3 to mimic the embryonic endocrine cell development explained above. We detailed this, as requested, in our revised manuscript.

In addition, as we described above, in every experiment and analysis we performed we included spheres (derived from pancreatic ducts) infected with control viruses (Ad-RFP or Ad-GFP) but otherwise identical conditions as with N, 4TF or 4TFM spheres. These controls served to monitor the possibility of spontaneous differentiation. Using the thorough analyses presented in our manuscript, we were unable to detect any sign of spontaneous endocrine cell differentiation in these controls. Therefore, we concluded that spontaneous conversion occurred at negligible frequency.

Is Pax6 already expressed in the MNP-adnovirus cultures? If so, why is more needed?

We thank the reviewer for this good question: Pax6 mRNA level in MNP spheres was less than 0.03% of levels detected in primary human islets. This finding promoted us to include this critical factor in our cocktail. To document this result, we have now included Pax6 qRT-PCR data in Figure 4–figure supplement 1 and in our revised manuscript.

The part on “We sought new methods to mature the cells” (my words) reads strangely. It's the same method, just extending the time frame, I believe, and I would simplify this text.

In addition to the extended time frame, we constructed new adenovirus permitting simultaneous expression of three factors (Pdx1, MafA, and Pax6) in a single virus, to increase the chance of these 3 factors being expressed in single cell. Therefore, we used only 2 viruses for 4TF (as opposed to 4 individual viruses for 4V). We clarified this in our revised manuscript.

Another comment here is that we revert to the MNP6 mixture (4TF), having just been told that Pdx1 can be removed without impact – why?

Even though exogenous PDX1 is not required for the conversion observed here, we found that exogenous PDX1 increased INS expression by two fold (Figure 4E). This motivated us to include PDX1 in our MAFA-PAX6-PDX1 virus (Ad-GFP-M6P) construction.

Figure 2–figure supplement 1Chas an incorrect y-axis. 10,000 percent to the log(base10) is 4, correct. This graph needs altering – why not just “fold” for cell number? Related to this, Discussion asserts 3,000-fold, but this is just once? Up to 3,000-fold, and more typically xx-fold? Why do the cultures suddenly go bad at G7 (see text) – what happens – sudden apoptosis? #48 seems to be continuing even at G7 – please amend this text.

We thank the reviewer for finding this error and we have amended the data presentation appropriately.

As was shown in Figure 2–figure supplement 1C, we observed only one sample reached to G7. We agree with the reviewer and changed “>3,000-fold” to “up to 3,200-fold”.

We found the cells stopped growing and therefore could not be passaged in spheres after G7. We revised the text as suggested to “ductal cell expansion was not achieved, and spheres were not formed” in our manuscript.

Does the 3-factor system (MNP) work in the authors' hands on mouse acini? And/or duct?

In preliminary studies, we have found that the MNP combination can induce insulin expression in cultured mouse ductal cells. If deemed important by the editors and reviewers, we are happy to include this unpublished data.

Does the CD133 separation method include centroacinar cells (CAC) or not? What is the photograph inFigure 1– ‘tip’ of duct dives out of plane of section before it is reached, and therefore the CAC cannot be seen in this panel? CAC could have a specific latency not seen in duct cells.

We also think this is an interesting point and included this in our discussion. Briefly, CD133 is expressed in CACs in human pancreas, so we cannot exclude the possibility that CACs are included in CD133 sorted cell fraction. Indeed this is highly likely. We were unable to test the presence of CACs in our sorted CD133+ fraction due to a lack of specific “human” CAC marker. However, the sphere-forming efficiency approached 1 in 5 cells in our assay, and the frequency of CACs is thought to be much lower than this.

The idea that lineage-tracing methods are hard to apply in human cells should be stated, as everything else hinges on numerical arguments on cleanliness of cell separations, etc.

We have included a sentence in the Discussion to cover this point.

chgA is an endocrine differentiation marker. This statement is meant to indicate that full-blown differentiation to the hormone-expressing state requires a substantial pulse of Ngn3. What about other hormones, even indicating non-pancreatic cell types?

We performed qRT-PCR with CCK (Cholecystokinin) and GAST (Gastrin) – hormones expressed in the intestine – in Neurog3-overexpressed spheres and were unable to measure any detectable mRNA. We included this in our manuscript.

A major point is the longevity of the pulse of Ngn3 and the other factors achieved with these methods, and the detection of a program that runs from the endogenous loci with or without the continuous presence of N, MNP, MNP6 (4TF, 4TFM). Would this method pulse the cells or not, and is continuous presence of some of the factors preventing their full differentiation to the most mature state?

Expression of the exogenous factors persisted after two weeks of maturation period (Figure 5–figure supplement 3) and at least 5 months, evidenced by the GFP-positive insulin-producing cells in transplanted mice (Figure 5–figure supplement 2A). Thus, the current method does not permit 'pulse' expression of the factors. Transgenes delivered by adenovirus do not generally persist long term (Zhou et al., 2008), especially in dividing cells. Persistent transgene expression noted in our study likely results from the known ability of Neurog3 to induce cell cycle exit (Miyatsuka et al., 2011), thereby preventing dilution of transgenes and transgene-encoded factors by cell division. This precluded tests to demonstrate independence of the transgene-encoded factors for ongoing maintenance of the converted-cell phenotypes observed. We also agree with the reviewer that it is possible the continuous presence of some of the factors may prevent full differentiation toward more physiologically-mature endocrine cells. However, we do not feel these limitations change the main conclusions of our study. We have included a discussion of these points in our revised manuscript.

Title of the section starting ‘Although ELISA studies readily detected Proinsulin production by IPCs…’ reads odd to me: ‘genetic conversion’ reads as if the genome of the adult duct cells is being altered in some manner.

We agree with this reviewer’s point. We changed the phrase “genetic conversion” to “conversion”.

Systematic removal of factors from MNP6 mixtures: Why can Pdx1 be removed without any impact?

It is unclear why exogenous PDX1 is not critical for ductal sphere conversion toward an endocrine fate. One possibility is that our findings indicate distinct transcription factor requirements of human ductal epithelium (compared to mouse acinar cells and human hepatocytes that require Pdx1 expression for conversion, like in Zhou et al., 2008). We included a discussion of this point in our revised manuscript.

Various ‘obvious’ markers were not tested, or the manuscript is incorrect in not showing such ‘easily pointed out’ questions. Pdx1 is produced, by immunofluorescence assay, within these induced beta cells? To normal levels? MafA/B, etc? Nkx6.1 was assessed, but the ‘dogmatic’ mature β-cell TF list should be addressed, at least.

We agree that MAFA and PDX1 are generally accepted adult β-cell markers in human. However, viral-expressed exogenous MAFA and PDX1 proteins from Ad-GFP-M6P adenovirus precluded us from detecting endogenous MAFA and PDX1 expression in IPCs. Based on this constraint, we used immunostaining to verify expression of other known ?-cell specific markers such as NKX6.1, IAPP, and PC1/3. We emphasized this point in our revised text.

It does seem difficult to follow 4V, 4TF, 4TFM, MNP. Seems as if there is a mixed descriptor used for the same manipulation, at least sometimes; I suggest simply checking for a way of making the text fully consistent throughout.

We have striven to achieve consistent use of these acronyms throughout the text in our revised manuscript.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#10160) of the Stanford University. All surgery was performed under anesthesia, and every effort was made to minimize suffering.

eLife is a non-profit organisation inspired by research funders and led by scientists. Our mission is to help scientists accelerate discovery by operating a platform for research communication that encourages and recognises the most responsible behaviours in science.eLife Sciences Publications, Ltd is a limited liability non-profit non-stock corporation incorporated in the State of Delaware, USA, with company number 5030732, and is registered in the UK with company number FC030576 and branch number BR015634 at the address:
eLife Sciences Publications, Ltd
1st Floor, 24 Hills Road
Cambridge CB2 1JP
UK